In general, biologically active polymeric substances such as proteins and DNAs cannot enter cells through cell membranes because they cannot pass through phospholipid bilayers. However, cell-penetrating peptides are known which can cross the cell membrane without the help of receptors or other molecules.
The cell-penetrating peptides, also called PTDs (protein transduction domains) or MTS (membrane-translocating sequences), can be associated or mixed with cargo such as proteins, DNAs, RNAs, etc. and deliver the cargo into cells through the cell membrane and also into the cytoplasm, cell organelles and nuclei (Endoh and Ohtsuki, 2010; Joliot and Prochiantz, 2004; Mogi and Kondo, 2010).
Tat is the first protein that has been found to penetrate the cell membrane during HIV-1 (human immunodeficiency virus-1) infection. The TAT peptide (YGRKKRRQRRR (SEQ ID NO: 15)) derived therefrom is the most frequently used and actively studied (Mann, D. A. et al., EMBO J 10: 1733-1739, 1991).
β-galactosidase, horseradish peroxidase, RNase A, the translocation domain of Pseudomonas exotoxin A (PE), etc. have been delivered into cells using the TAT peptide for researches of their functions and intracellular localization (Fawell, S. et al., PNAS 91: 664-668, 1994). It has been found that the TAT peptide enters the cells by interacting with heparan sulfate on the cell membrane, followed by endocytosis wherein lipid rafts are involved (Jehangir S. W. et al., Nature Med 10: 310-315, 2004).
In addition, penetratin (Antp), consisting of 16 amino acids, which is derived from Antennapedia homeoprotein and an essential transcription factor in the development of a fruit fly, cell-penetrating peptide VP22 which is derived from the VP22 protein expressed by HSV-1 (herpes simplex virus type 1), transportan, consisting of 27 amino acids, which has been artificially synthesized, polyarginine which has been obtained by artificially repeating the arginine residues expected to play the most important role in cell-penetrating peptides, etc. are well known as cell-penetrating peptides.
These existing cell-penetrating peptides may cause side effects such as immune responses when used in human bodies because they are derived from the proteins of viruses such as HIV-1, derived from the proteins expressed by other species such as fruit fly or artificially synthesized based on the amino acid sequence analysis of the previously known cell-penetrating peptides.
In addition, they are more likely to cause unwanted immune responses because they consist of relatively long amino acid chains. And, the efficiency of linking with biologically active substances to be delivered into cells is often low because they may affect the structure and function of the proteins to be delivered.